Saudi Journal of Medical and Pharmaceutical Sciences

Abbreviated Key Title: Saudi J Med Pharm Sci

ISSN 2413-4929 (Print) |ISSN 2413-4910 (Online)
Scholars Middle East Publishers, Dubai, United Arab Emirates
Journal homepage: http://scholarsmepub.com/sjmps/

Original Research Article

Phytopharmacological Study of Piper Betle Leaf

Nishant M Patel*, Darshankumar D. Jain, Hemant P. Suryawanshi, Sunil P Pawar
P.S.G.V.P.M’s College of Pharmacy, Shahada (MS), India

DOI: 10.36348/sjmps.2019.v05i11.008 | Received: 07.11.2019 | Accepted: 14.11.2019 | Published: 16.11.2019

*Corresponding author: Mr. Nishant Mahendra Patel

Abstract

Piper betle L. is an evergreen perennial dioecious creeper and it is native to Malaysia. It is commonly used in our
day to day life. In present investigation an attempt has been made for the pharmacognostic standardization and
phytochemical evaluation of Piper betle L. leaves. The pharmacognostic evaluation comprises of detailed
morphology, powdered microscopy, quantitative microscopy and physical constants such as extractive and ash
values. The leaves extract were subjected to preliminary phytochemical screening. The data obtained in present
study will serve as valuable tool for identification, authentication and detection of adulterants standardization and
quality control of the drug. In this research work the anthelmintic property of leaves extract of Piper betle L.
belonging to family Piperaceae was studied for anthelmintic activity against Pheretima Posthuma (Indian
earthworm). The leaves powder of Piper betle L. was subjected to successive extraction by maceration using
solvents like Aqueous, Acetone + Aqueous and Pet. Ether. Four concentrations (25, 50, 75 and 100 mg / ml) of
different extract of leaves were studied in a bioassay, which involved the determination of time of paralysis and
time of death of the worms. 100 mg/ml conc. of Aqueous + Acetone extract of leaves of Piper betle L. reveal
considerable anthelmintic activity as compared to other two concentrations and piperazine citrate (10 mg/ml).
Piperazine citrate and saline water were included in the assay as standard reference drug and control, respectively.
Keywords: Piper betle, Phytochemistry, Anthelmintic activity, Pheretima Posthuma etc.
Copyright @ 2019: This is an open-access article distributed under the terms of the Creative Commons Attribution license which permits unrestricted
use, distribution, and reproduction in any medium for non-commercial use (NonCommercial, or CC-BY-NC) provided the original author and source
are credited.

INTRODUCTION  Genus : Piper

Piper betle L. (Piperaceae) is an evergreen  Species : Piper betle L
perennial dioecious creeper and It is commonly known  Division : Magnoliphyta
as ‘Paan’ or ‘Nagvalli’[1]. From ancient time betel are
chewed along with areca nut, slaked lime, cardamom Local names [11]
and clove in many Asian countries and offering betel  Marathi : Nagabael
morsel (Pan-Supari) to guests in Indian subcontinent is  Hindi : Paan
a common courtesy [2]. This plant is extensively grown
 Sanskrit : Tambool
in Bangladesh, India, Sri Lanka, Malaysia, Thailand,
Taiwan and other Southeast Asian countries [3].  Malayalam : Vettila
Recently, the medicinal uses and properties of areca nut  Kannada : Eleballi
were investigated. It has antimicrobial [4], antifertility  Gujarati : Nagarbael
[5], antihyperglycemic [6], antidermatophytic [7]  Tamil : Vetrilai
antinociceptive [8] analgesic activity [9]. In spite of all  Bengali : Paan
these medicinal values of Piper betle L, its chronic  Telgu : Nagballi
consumption or chewing may cause several adverse  English : Betel-vine
effects including carcinogenesis.
Description of Plant
Taxonomical classification [10] Is a perennial dioeciously creeper. Stems are
 Kingdom : Plantae semi woody, climbing by means of short adventitious
 Order : Piperales roots. Leaves are 10-20 cm long, broadly ovate, slightly
 Family : Piperaceae cordate and often unequal at the base, shortly
 Subfamily : Piperoideae acuminate, glabrous, and glaucous on sides, bright
green or yellowish, petiole stout 2.0-2.5 cm long. The
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Male spikes are cylindrical dense. Female spikes 2.5-
5.0cm long, pendulous. Fruits rarely produced, often
sunk in the fleshy spike, forming nodule-like structures.
The branches of the plant are swollen at the
nodes. The plant has alternate, heart-shaped, smooth,
shining and long-stalked leaves, with pointed apex [12].
Fig-1: Creeper of Piper betel L [13]
Chemical Constituents
Piper betle L. leaf has been described to have
piperol-A, piperol-B, methyl piper betlol and they also
have been isolated. Piper betel leaves have an essential
oil composing of terpinen-4-ol, safrole,
allylpyrocatecholmonoacetate, eugenol, eugenyl
acetate, hydroxyl chavicol, eugenol, piper betol and the
betel oil contains cadinenecarvacrol, allyl catechol,
chavicol, pcymene, caryophyllene, chavibetol, cineole
and estragolas the major components [14-16].
Cultivation and collection [10, 11]
Propagation
 Propagation is easy by root division or
cuttings. Betel requires well-drained fertile soil
semi-shade position.
Waterlogged, saline and alkali soils are
unsuitable for its cultivation.
Cultivation
 First of all prepare a garden called a barouj in
which to grow betel. The barouj is fenced
with bamboo sticks and coconut leaves.
 The soil is plowed into furrows of 10 to 15
meter length, 75 cm in width and 75 cm in
depth.
 Oil cakes, manure and leaves are thoroughly
incorporated with the topsoil of the furrows
and wood ash. The cuttings are planted at the
beginning of the monsoon season.
 Proper shade and irrigation are essential for the
successful cultivation of this crop. Betel needs
constantly moist soil, but there should not be
© 2019 |Published by Scholars Middle East Publishers, Dubai, United Arab Emirates
excessive moisture. Irrigation is frequent and
light and standing water should not remain for
more than half an hour.
Collection
 In 3 to 6 months the vines reach 150 to 180
centimeters in height and they will branch.
 Harvest begins, with the farmer plucking the
leaf and its petiole with his right thumb.
 The harvest lasts 15 days to one month.
MATERIAL AND METHODS
Procurement of Plant Material
The leaves of Piper betle L. have been
collected from the local region of Shahada. The plant is
authenticated by Dr. Santosh Tayade, Dept. of Botany,
ASC College, Lonkheda, Shahada, Nandurbar (M.S).
The voucher specimen has been preserved in the
laboratory for future reference in the Dept. of
Pharmacognosy and Phytochemistry, COP, Shahada.
The collected plant material was air dried and
used for the study of macroscopic and microscopic
characters. Finally dried leaves were subjected to size
reduction to get coarse powder and then passed through
sieve no. 40 to get uniform powder. Then uniform
powder was subjected for the determination of ash
values, extractive values, and loss on drying and
phytochemical constituents.
Pharmacognostic evaluation
Organoleptic Evaluation
In organoleptic evaluation, various sensory
parameters such as color, odour, taste, shape, apex,
venation, and texture of the leaves were observed and
shown in Table No.1.
Microscopical Evaluation
T. S. of Piper betle L. leaves
The T.S. of the Piper betle L. leaves were
taken to observe microscopic characteristics
Characteristic are shown in microscopic pictures as Fig.
2.
Powder Analysis
To a little quantity of powder taken onto a
microscopic slide, 1 – 2 drops of 0.1% phloroglucinol
solution and a drop of concentrated hydrochloric acid
were added, covered with a cover slip and observed
under microscope with 10 × 10 magnification. The
characteristic features of the powder like fiber,
trichome, collenchyma cells, etc. were recorded using
standard techniques. The characteristic are shown in
microscopic pictures as Fig. 3, 4, and 5 respectively.
Physical evaluation
In physical evaluation, ash values viz., total
ash, acid insoluble ash and water soluble ash, and
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extractive values viz., water soluble extractive and pet.
Ether extractive values were determined.
Determination of ash values [17]
Total Ash Value
Accurately weighed (2 g) of air-dried Piper
betle L. leaves powder was taken in a silica dish and
incinerated at a temperature not exceeding 450°C until
free from carbon. The resultant ash was cooled and
weighed. The % of ash was calculated with reference to
the air dried drug.
Acid Insoluble Ash
The total ash obtained from (2 g) of Piper
betle L. leaves powder was boiled for 5 minutes with 25
ml of dilute HCl and the insoluble matter was collected
on an ash less filter paper. It was washed with hot
water, ignited and weighed. The % of acid insoluble ash
was calculated with reference to the air dried drug.
Water Soluble Ash
The total ash obtained from (2 g) Piper betle
L. leaves powder was boiled for 5 minutes with 25 ml
of water, the insoluble matter was collected on an ash
less filter paper, washed with hot water, and ignited for
15 min at a temperature not exceeding 450°C. The
weight of insoluble matter was subtracted from the
weight of the ash, the difference in weight represent the
water-soluble ash was calculated with reference to the
air-dried drug.
Extractive values [18]
Pet. ether (40 - 60°C) soluble extractive value
Accurately weighed (5 g) of leaves powder of
Piper betle L. was macerated with 100 ml of Petroleum
ether in a closed flask, shaking frequently during the
first 6 hours and allowed to stand for 18 hours.
Thereafter, it was filtered rapidly taking precaution
against loss of Petroleum ether. Evaporated 25 ml of
filtrate to dryness in a tarred flat bottom shallow dish
dried at 105°C and weighed and % Petroleum ether
soluble extractive was calculated with reference to the
leaves.
Water-soluble extractive value
Accurately weighed (5 g) of leaves powder of
Piper betle L. was added to 50 ml boiled water at 80°C
in a stoppered flask. It was then shaken well and
allowed to stand for 10 min so as to cool it and filtered.
5ml of filtrate was transferred to an evaporating dish,
which was 7.5 cm in diameter, the solvent was
evaporated on water bath, allowed to dry for 30
minutes, finally dried in an oven for 2 hrs At 100°C
and residue was weighed % of water soluble extractive
value was calculated with reference to the air dried
drug.
© 2019 |Published by Scholars Middle East Publishers, Dubai, United Arab Emirates
Loss on drying [19]
Accurately weighed (2 g) quantity of leaves
powder of Piper betle L. was taken in tarred glass bottle
and initial weight was taken. The sample was heated at
105°C in an oven and weighed. This procedure was
repeated until a constant weight was obtained. The
moisture content of the sample was calculated with
reference to air-dried drug.
Preliminary phytochemical screening [20]
The leaves powder are subjected to successive
extraction by maceration by using water + acetone, the
extracts where evaporated to dryness. The dried extracts
were weighed. The % yield of leaves of Piper betle L.
after maceration was found to be max of 8.12.
Phytoconstituents present in extract of leaves of
Piper betle L
The extract were used for preliminary
phytochemical with a chemical tests viz., Molisch’s,
fehling’s, Benedict’s and Barfoed’s tests for
carbohydrates; Ninhydrin’s test for amino acids;
Salkowski and Libermann-Burchard’s reaction for
steroids; Foam test for saponin glycosides;
Dragendorff’s, Mayer’s, Hager’s and Wagner’s tests for
alkaloids; and ferric chloride, lead acetate, potassium
dichromate, and dilute iodine tests for tannins, shinoda
test for flavonoids The results are given below in Table
No.5.
Pharmacological evaluation
Anthelmintic activity
The anthelmintic assay was carried as per the
method of Ajaiyeoba et al. with necessary modification
[21]. The assay was performed on adult Indian
earthworm, due to its anatomical and physiological
resemble with the intestinal round worm parasite of
human being [22, 23]. Because of easy availability,
earthworm has been used widely for initial evaluation
of anthelmintic compound in vitro [24]. 25 ml of
formulation containing different concentration of crude
drug aqueous + acetone extract (25, 50, 75,100 mg/ml
in dist. water) were prepared and 4 worms of same type
were placed in it. Time for paralysis was noted when no
movement of any sort could be observed except when
the worms were shaken vigorously. Time for death of
worms were recorded after ascertaining that worms
neither move when shaken vigorously nor when dipped
in warm water (50°C). Piperazine citrate (10 mg/ml)
was used as reference standard while saline water as
control.
MATERIALS AND METHODS
Plant Material
The fresh leaves of Piper betle L. have been
collected from the local area at Shahada and
authentified by Dr. Santosh. K. Tayade, HOD of
Botany, ASC College, Lonkheda, Shahada.
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Worms
Indian earthworms (Pheretima Posthuma) Drugs and chemicals
were used to study anthelmintic activity. The  Piperazine citrate.
earthworms were collected from moist soil at local  Saline solution.
area at Taloda, Nandurbar. The average size of
earthworm was 6 – 8 cm. All worms were washed with RESULTS
normal saline,and kept in beakers containing normal In the present study leaves of Piper betle L.
saline separately. was evaluated for its pharmacognostic, phytochemical
and pharmacological aspects which revealed the
Preparation of Extracts following results.
Collected fresh leaves of Piper betle L. were
dried and crushed to coarse powder and pass it through Pharmacognostic evaluation
sieve no 40 to get fine powder and subject it to Organoleptic Evaluation
maceration with Aqueous + Acetone, and Pet. ether The results of organoleptic evaluations were
then dried it by using evaporator and then extract was given in Table No. 1.
subjected to preliminary phytochemical testing [25-27].

Table-1: Organoleptic/Macroscopic characteristics of leaves of Piper betle L.

Sr. No. Parameters Observation of Leaf
1. Colour Yellowish Green -Dark Green
2. Odour Characteristic and pleasant
3. Taste Sweet to Pungent
4. Shape Heart shape
5. Texture Glossy, smooth
7. Venation Reticulate
8 Apex Pointed

Microscopical Evaluation
Transverse Section of Piper betle L. leaves

Fig-2: Transverse section of the leaves of Piper betle L.

The transverse section of the leaves of Piper Table-2: Microscopic characteristic of transverse
betle L. (Fig. 2) shows the typical microscopic section of leaves of Piper betle L.
characteristics like epidermis, collenchyma, xylem, Sr. No. Observation of T. S of leaf
phloem, schizogenous oil cavity, ground tissues, 1. Epidermis
secretory cell, trichome, etc. The characteristic are 2. Collenchyma
shown Table No. 2. 3. Xylem
4. Phloem
5. Schizogenous oil cavity
6. Ground tissues
7. Secretory cell
8. Trichome

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Powder Analysis
The powder of the leaves of Piper betle L.
shows the typical microscopic characteristics like
fibres, trichome and collenchyma cell etc. The
characteristic are shown Table No. 3 and Fig. 3, 4 and 5
respectively.

Fig-5: Collenchyma cells

Fig-3: Fiber Table-3: Microscopic characteristic of for leaves

powder of Piper betle L
Sr. No. Observation of Piper betle L. powder
1. Fibers
2. Trichome
3. Collenchyma cells
Fig-4: Trichome
Physical evaluation
 Ash Values: The total cash value, acid insoluble
ash value and water soluble ash value was found to
be 3.1 %w/w, 1.2 %w/w and 1.9 %w/w
respectively as shown in Table No.4.
 Extractive Values: The water soluble and
Petroleum ether soluble extractive values were
found to be 3.9 %w/w and 4.5 %w/w respectively
as shown in Table No.4.
 Loss on Drying: The loss on drying was found to
be 0.80%w/w as shown in Table No.4.

Table-4: Physical constants for leaves powder of Piper betle L

Sr. No. Physical Constants Results
Ash Values
 Total Ash 3.1 %w/w
1.
 Acid Insoluble Ash 1.2 %w/w
 Water Soluble Ash 1.9 %w/w
Extractive Values
2. A) Water soluble extractive 3.9 %w/w
B) Petroleum ether soluble extractive 4.5 %w/w
3. Loss on Drying 0.80 %w/w

Preliminary phytochemical screening Phytoconstituents present in aqueous + acetone

The percentage yield of leaves of Piper betle
L. after maceration was found to be max 8.12 % as
compared to other extracts
extract of Piper betle L. leaves
In the present investigation the extract of plant
was analyzed for the presence of alkaloids,
carbohydrates, glycosides, amino acids, steroids,
tannins and flavonoids using standard procedures. The
preliminary phytochemical investigation showed the
presence of alkaloids, carbohydrates, saponins, tannins,
steroids, glycosides, flavonoids and amino acids as
shown in Table No. 5.

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Table-5: Data showing the presence of phytoconstituents present in aqueous + acetone extract of Piper betle L.
leaves
Sr. No Phytoconstituents Aqueous + Acetone extract
1 Alkaloids Present
A Dragendorff’s Reagent +
B Hager’s Reagent -
C Wagner’s Reagent +
D Mayer’s Reagent +
2 Carbohydrate Present
A Molisch’s test +
B Benedict’s test +
C Fehling’s test +
3 Saponins Present
A Foam test +
4 Tannins Present
5 Steroids Present
A Salkowski test +
B Libermann-Burchard’s test +
6 Glycosides Present
7 Flavonoids Present
A Shinoda test +
B Lead acetate test +
8 Amino acids Present
A Ninhydrin’s test +

Pharmacological evaluation
Anthelmintic activity

Table-6: Anthelmintic activity of Piper betle L. leaves extracts on Indian earthworms (Pheretima Posthuma)
Indian Earthworm
Conc.
Extracts (Pheretima Posthuma)
(in mg/ml)
Sr. No. Time of Paralysis in min (P) Time of Death in min (D)
25 25.90 ± 1.22 101.22 ± 2.50
50 12.84 ± 1.30 41.05 ± 2.30
1 Aqueous
75 4.20 ± 1.25 12.78 ± 1.20
100 1.90 ± 0.55 11.15 ± 1.50
25 22.50 ± 1.20 98.11 ± 2.0
50 10.78 ± 1.00 38.31 ± 2.15
2 Aqueous + Acetone
75 2.50 ± 1.35 9.80 ± 1.15
100 1.00 ± 0.66 8.20 ± 1.20
25 27.50 ± 1.15 102.30 ± 2.40
Petroleum Ether 50 13.11 ± 1.60 42.50 ± 2.25
3
75 5.15 ± 1.30 13.50 ± 1.65
100 2.20 ± 0.50 12.00 ± 1.40
4 Control (Normal Saline) - - -
5 Standard (Piperazine citrate) 10 9.12 ± 0.75 12.95± 1.29

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Graph-1: Anthelmintic activity of Piper betle L.
leaves extracts on Indian earthworm (Pheretima
Posthuma)
Aq. E = Aqueous Extract, AA. E = Aqueous + Acetone
Extract, PE. E = Petroleum Ether Extract, Std =
Standard, (P) = Paralysis Time, (D) = Death Time
RESULT
From the above observations, higher
concentration of extract produced paralytic effect much
earlier and the time to death was shorter for all worms.
All the extracts showed anthelmintic activity but
Aqueous + Acetone extract showed better anthelmintic
activity than other extracts, in dose-dependent manner
giving shortest time of paralysis (P) and death (D) with
100 mg/ml concentration. Evaluation of anthelmintic
activity was compared with reference standard
Piperazine citrate.
From Table No. 6, it is observed that 100
mg/ml conc. of leaves of Piper betle L. shown potent
anthelmintic activity while 75, 50 and 25 mg/ml conc.
taken more time for paralysis as well as death of
worms. The results are compared with the standard drug
Piperazine citrate. For Aqueous + Acetone extract the
time of paralysis (P) was found to be 22.50, 10.78, 2.50
and 1.00 min. at conc. of 25, 50, 75 and 100 mg/ml
respectively when compared to standard i.e. Piperazine
citrate (9.12 min.) at the conc. of 10 mg/ml and to
normal saline as control. While the time of death (D)
was found to be 98.11, 38.31, 9.80 and 8.20 min. for
Aqueous + Acetone extract, at conc. of 25, 50, 75 and
100 mg/ml respectively when compared to standard i.e.
Piperazine citrate (12.95 min.) at the conc. of 10 mg/ml
and to normal saline as control. The results obtained
indicate that Aqueous + Acetone extract of Piper betel
L. leaves shown significant anthelmintic activity at
conc. of 100 mg/ml against Pheretima Posthuma. The
results of anthelmintic activity are shown in Table No.
6 and the graphical representations of results were
shown in Graph No.1.
DISCUSSION
Piper betle L. is widely use in the traditional
system of medicine for the treatment of number of
diseases. The results of these investigations would
© 2019 |Published by Scholars Middle East Publishers, Dubai, United Arab Emirates
helpful for proper identification, collection and
investigation of the plant. The parameters determined in
quantitative microscopy can be helpful to differentiated
closely related species. The presence of various
phytoconstituents can serve to treat diseases by using
various pharmacological activities. Physical standards
may be used to determine the quality of this plant in
future investigation. It will also helpful to carry out
further research and revalidation of its use in traditional
system in medicine.
It also gives anthelmintic activity and the
experimental evidence obtained in the laboratory model
could provide a rationale for the traditional use of this
plant as anthelmintic. The plant may be further explored
for its phytochemical profile to recognize the active
constituent accountable for anthelmintic activity.
Further studies using in vivo models are required to
carry out and establish the effectiveness and
pharmacological rationale for the use of Piper betle L.
leaves as an anthelmintic drug. The drug can be further
explored for the isolation and characterization of the
active constituents responsible for anthelmintic activity.
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